Methods for reactivating genes on the inactive X chromosome

Abstract

Methods for reactivating genes on the inactive X chromosome that include administering one or both of a DNA methyltransferase (DNMT) Inhibitor and/or a topoisomerase inhibitor, e.g., etoposide and/or 5-azacytidine (aza), optionally in combination with an inhibitor of XIST RNA and/or an Xist-interacting protein, e.g., a chromatin-modifying protein, e.g., a small molecule or an inhibitory nucleic acid (such as a small inhibitory RNA (siRNAs) or antisense oligonucleotide (ASO)) that targets XIST RNA and/or a gene encoding an Xist-interacting protein, e.g., a chromatin-modifying protein.

What is claimed is: 
     
       1. A method of activating an inactive X-linked allele in a cell of a female heterozygous mammalian subject, the method comprising administering to the cell 
       (i) a DNA methyltransferase (DNMT) Inhibitor; and 
       (ii) an inhibitory nucleic acid targeting Xist RNA, wherein the inhibitory nucleic acid is complementary to at least 8 consecutive nucleotides of XIST RNA. 
     
     
       2. The method of  claim 1 , wherein the X-linked allele is an epigenetically silenced or hypomorphic allele on the active X-chromosome. 
     
     
       3. The method of  claim 1 , wherein the subject has an X-linked disorder selected from the group consisting of Rett Syndrome, CDKL5 Syndrome, and Fragile X Syndrome, and the DNMT Inhibitor and the inhibitory nucleic acid targeting Xist RNA are administered in an amount sufficient to alleviate or partially arrest at least one clinical manifestation of the X-linked disorder or its complications. 
     
     
       4. The method of  claim 1 , wherein the inactive X-linked allele is associated with an X-linked disorder selected from the group consisting of Rett Syndrome, CDKL5 Syndrome, and Fragile X Syndrome, and the DNMT Inhibitor and the inhibitory nucleic acid targeting Xist RNA are administered in an amount sufficient to alleviate or partially arrest at least one clinical manifestation of the X-linked disorder or its complications. 
     
     
       5. The method of  claim 1 , wherein the cell is in a living subject. 
     
     
       6. The method of  claim 1 , wherein the inhibitory nucleic acid comprises at least one ribonucleotide, at least one deoxyribonucleotide; or at least one bridged nucleotide. 
     
     
       7. The method of  claim 6 , wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide. 
     
     
       8. The method of  claim 1 , wherein the inhibitory nucleic acid does not comprise three or more consecutive guanosine nucleotides or does not comprise four or more consecutive guanosine nucleotides. 
     
     
       9. The method of  claim 1 , wherein the inhibitory nucleic acid is 8 to 30 nucleotides in length. 
     
     
       10. The method of  claim 1 , wherein at least one nucleotide of the inhibitory nucleic acid is a nucleotide analogue. 
     
     
       11. The method of  claim 1 , wherein at least one nucleotide of the inhibitory nucleic acid comprises a 2′ O-methyl. 
     
     
       12. The method of  claim 1 , wherein one or more of the nucleotides of the inhibitory nucleic acid comprise 2′-fluoro-deoxyribonucleotides and/or 2′-O-methyl nucleotides. 
     
     
       13. The method of  claim 1 , wherein the nucleotides of the inhibitory nucleic acid comprise a phosphorothioate internucleotide linkage between at least two nucleotides, or between all nucleotides. 
     
     
       14. The method of  claim 1 , wherein the subject is a human. 
     
     
       15. The method of  claim 1 , wherein the inactive X-linked allele was inactivated by Xist RNA. 
     
     
       16. The method of  claim 1 , wherein the inhibitory nucleic acid is complementary to at least 10 consecutive nucleotides of XIST RNA. 
     
     
       17. The method of  claim 1 , wherein the inhibitory nucleic acid is complementary to at least 12 consecutive nucleotides of XIST RNA. 
     
     
       18. The method of  claim 1 , wherein the inhibitory nucleic acid is complementary to at least 15 consecutive nucleotides of XIST RNA.